The vast majority of strokes are ischemic/thrombotic in nature. There is no effective therapy for stroke. Our earlier work showed early post-ischemic neuroprotection by activated protein C (APC), a protease with anti- thrombotic, anti-apoptotic and anti-inflammatory activities, as well as that protease activated receptor 1 (PAR1) and endothelial protein C receptor (EPCR) on brain endothelial cells (BEC), and PAR1 and PAR3 on neurons mediate APC's neuroprotection and inhibition of mitochondria-dependent and death receptor-dependent apoptosis. This work led to a Phase 1/2a clinical trial with APC (Xigris) for acute ischemic stroke with a 6 h therapeutic window (APCAST). Our recent pilot studies indicated that recombinant wtAPC was neuroprotective when given 24 h after ischemia onset suggesting a much larger therapeutic window for APC. Late post- ischemic 6-72 and 72-144 h multi-dosing therapies with wtAPC stimulated neovascularization and neurogenesis. Multi-dosing therapy with 3K3A-APC, an APC mutant with reduced anticoagulant activity, was neuroprotective when initiated 12 h after ischemia onset and stimulated neurogenesis. The cytoprotective- selective 5A-APC mutant with < 10% anticoagulant activity, but not the anticoagulant-selective E149A-APC with 400% increased anticoagulant activity and no cytoprotective activity, was neuroprotective after stroke in mice and increased post-ischemic in vivo dendritic spine formation. Our pilot data show human 3K3A-APC mediates angiogenesis from human BEC and neurogenesis from human neuronal progenitor cells (NPC) in processes that require EPCR, PAR1 and sphingosine 1-phosphate receptor 1 (S1P1), and PAR1, PAR3 and S1P1, respectively. Our central hypothesis is that late therapy with recombinant wtAPC and its cytoprotective- selective 5A-APC mutant, but not the anticoagulant-selective E149A-APC mutant, is neuroprotective and promotes brain regeneration after ischemic/thrombotic stroke by modifying neuronal plasticity, vascular remodeling and stroke-induced neurogenesis through PAR1, EPCR and S1P1 on BEC, and PAR1, PAR3 and S1P1 on NPC. Recombinant murine wtAPC, 5A-APC and E149A-APC will be studied in murine stroke models and human APCs on human BEC and NPC. We propose to test first safety and efficacy of late therapies with wtAPC, 5A-APC and E149A-APC using models of cortical ischemic stroke or photothrombotic ministroke in control mice (aim 1); and in mice with PAR1-4 deletions, EPCR depletion and neural-specific loss of S1P1 (aim 2). The effects of APCs on in vivo neuronal plasticity, vascular remodeling (aim 1), neovascularization (aim 3) and neurogenesis (aim 4) and the receptors requirements for these APC-mediated processes in vivo will be determined. Human BEC (aim 3) and NPC (aim 4) will be used to determine in vitro receptors requirements and downstream Akt targets critical for APC's in vitro angiogenesis and neurogenesis. The proposed studies will bridge the field of ischemic stroke to the field of protein C with an emphasis on late neuroprotection and brain repair therapies that is promising for translation from bench to bedside. PUBLIC HEALTH RELEVANCE: Stroke is a major cause of morbidity and mortality for Americans caused by an acute block in blood supply to the brain resulting often in an irreversible brain damage. There is no effective therapy for stroke today. The proposed studies should help develop the rationale for clinical trials of activated protein C (APC) and its analogs with a substantially extended therapeutic window for stroke to help stroke patients over an extended period of time after stroke occurred with two primary goals: (1) to protect brain during periods of abnormally low blood supply; and (2) to support regeneration and brain repair process during a recovery phase after stroke.